Androgenic and antiandrogenic activities in water and sediment samples from the river Lambro, Italy, detected by yeast androgen screen and chemical analyses

Chemosphere 67 (2007) 1080–1087 www.elsevier.com/locate/chemosphere

Androgenic and antiandrogenic activities in water and sediment samples from the river Lambro, Italy, detected by yeast androgen screen and chemical analyses Ralph Urbatzka a, Anne van Cauwenberge b, Silvia Maggioni d, Luigi Vigano` c, Alberta Mandich e, Emilio Benfenati d, Ilka Lutz a, Werner Kloas a,f,* a

Department of Inland Fisheries, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Berlin, Germany b Institut Provincial d’Hygie`ne et de Bacte´riologie, Mons, Belgium c Water Research Institute, National Council of Research (IRSA-CNR), Brugherio (Milan), Italy d Mario Negri Institute, Laboratory of Environmental Chemistry and Toxicology, Milan, Italy e Department of Environmental, Experimental and Applied Biology, University of Genoa, Italy f Department of Endocrinology, Institute of Biology, Humboldt-University, Berlin, Germany Received 5 July 2006; received in revised form 31 October 2006; accepted 24 November 2006 Available online 17 January 2007

Abstract The river Lambro is the most polluted tributary of the river Po in North Italy and was chosen as a representative water course discharging industrialized areas. Water and sediment samples of the river Lambro were investigated regarding the presence of endocrine disrupting compounds. A combined procedure was used consisting of solid-phase extraction and HPLC based fractionation of samples, of screening for (anti)androgenic activity using the yeast androgen screen (YAS) and of chemical analysis using HPLC–MS/MS and GC– MS. Androgenic and antiandrogenic activities were found in specific fractions of the water and sediment while the total extracts showed antiandrogenic activity only. The chemical analysis of the fractions and total extracts with antiandrogenic activities revealed the presence of compounds with suspected antiandrogenic potency such as bisphenol A, iprodione, nonylphenol, p,p 0 -DDE and tert-octylphenol but other unknown compounds contributed mainly to the observed antiandrogenic activities. The antiandrogenic load of the river Lambro ranged between 1.34 and 17.1 lM flutamide-equivalents and may pose a risk to aquatic environments. Future screenings for EDC in the environment that have the potential to interfere with reproduction of aquatic organisms should be extended to different modes of actions including (anti)androgenic ones.  2006 Elsevier Ltd. All rights reserved. Keywords: Endocrine disruption; YAS; Environment; HPLC–MS/MS; GC–MS

1. Introduction Many chemicals released into the environment have been shown to interfere with the endocrine systems of different organisms and are called endocrine disrupting com*

Corresponding author. Address: Department of Inland Fisheries, Leibniz-Institute of Freshwater Biology and Inland Fisheries, Mu¨ggelseedamm 310, 12587 Berlin, Germany. Tel.: +49 30 64181630; fax: +49 30 64181799. E-mail address: [email protected] (W. Kloas). 0045-6535/$ - see front matter  2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2006.11.041

pounds (EDC). Most attention in the field of endocrine disruption has been related to the estrogenic potential of chemicals. Several studies demonstrated estrogenic activity of single chemicals (Routledge and Sumpter, 1996; Folmar et al., 2002) and of environmental samples representing a mixture of synthetic and natural EDC (Cargouet et al., 2004; Pawlowski et al., 2004; Petrovic et al., 2004). In contrast, androgenic and antiandrogenic activities of chemicals only recently came into focus. The interaction of (anti)androgenic chemicals with the androgen receptor may influence the normal sexual differentiation of males

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and may lead to male reproductive disorders (Kelce and Wilson, 1997). Especially the antiandrogenic activity of chemicals is hypothesized to be related to adverse effects of male reproduction (Fisher, 2004). In vitro assays have been established to screen for androgenic and antiandrogenic activity of chemicals. Androgenic activity in the environment was reported in pulp and paper mill effluents (Svenson and Allard, 2004), and in river water and effluents from wastewater treatment plants (Thomas et al., 2002; Blankvoort et al., 2005). While androgenic activity in rivers may be in part a result of microbial degradation of phytosterols to progesterons and then to androgens (Jenkins et al., 2004), antiandrogenic activity is mostly known from anthropogenic chemicals. Several compounds have been described besides other modes of actions to act as antiandrogens, e.g. the fungicide vinclozoline (Sohoni and Sumpter, 1998), the insecticide fenthion (Kitamura et al., 2003), the herbicide linuron (Lambright et al., 2000), the industrial compounds bisphenol A (BPA) and nonylphenol (Lee et al., 2003), the insecticide fenitrothion (Tamura et al., 2001) and the fungicide prochloraz (Vinggaard et al., 2002). Antiandrogenic activity also was detected in diesel exhaust particles (Kizu et al., 2003) and in components of sunscreens (Suzuki et al., 2005). Despite the knowledge about antiandrogenic potential of chemicals is increasing, the presence and the fate of antiandrogenic chemicals in the environment are not well established. The river Lambro, a major confluent to the river Po in Italy, was chosen as representative water course discharging industrialized areas. The river Lambro is suspected to be contaminated with EDC since intersex in fish was observed downstream of the confluent of the river Lambro (Vigano et al., 2001). For this reason water and sediment samples of the river Lambro in Italy were taken, extracted and fractionated. The yeast androgen screen (YAS) is suitable to investigate agonists as well as antagonists of the androgen receptor (Sohoni and Sumpter, 1998) and was used for the detection of androgenic as well as of antiandrogenic activities in the water and sediment samples of the river Lambro. In parallel, chemical analyses were applied to identify compounds with known antiandrogenic activity in the same samples. 2. Materials and methods 2.1. Chemicals and samples Methyldihydrotestosterone (MDHT) and flutamide (FLU) were from Sigma Aldrich (Taufkirchen, Germany). All chemicals needed for the media preparation were ordered by Sigma Aldrich except for chlorophenolredgalactoside CPRG (Roche Diagnostics, Mannheim, Germany). Ethanol (EtOH, 99.9%, Roth, Karlsruhe, Germany) was used as solvent for the standard chemicals as well as for the preparation of the lyophilized samples of water and sediment from the river Lambro, Italy. The water and sediment samples (the lyophilized product was

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derived from original volume 1 l water or 0.5 g sediment) were dissolved in 1 ml EtOH. 2.2. Extraction and fractionation of water and sediment samples A water sample of 10 l was collected in a glass vessel from the river Lambro, just before its confluence with the river Po. After filtration through glass fibre filters (Whatman GF-F) and acidification to pH 3 (HCl; Aristar, BDH, Poole, UK), the water sample was passed through Oasis cartridges (200 mg Oasis HLB glass cartridge; Waters, Milford, MA, USA), at a flow rate of approx. 6– 7 ml min1. Each cartridge was eluted with 6 ml of 10% methanol (MeOH) in tert-butyl methyl ether (Sigma Aldrich, Milan, Italy). All the eluates were pooled and reduced in volume by rotary evaporation, then split into two equivalent subsamples. The first one was in turn split into smaller aliquots, and dried under a flow of nitrogen and dim light conditions. These aliquots, each equivalent to 1 l of the original river water sample, were tested in vitro as whole water extracts. The second subsample was reduced to 1 ml and used for a HPLC based fractionation procedure. A HPLC Varian 9012Q (Varian Inc., Palo Alto, CA, USA) equipped with a C18 semi-preparative column (250 · 10 mm, Luna 10u C18(2) 100A, Phenomenonex, Torrance, CA, USA) and a C18 guard cartridge (10 · 10 mm, Phenomenonex, Torrance, CA, USA), was used to fraction the second subsample of the river water extract. The gradient used started with 2% MeOH and increased to 95% MeOH within 43 min, then to 100% MeOH in 7 min, followed by 10 min at 100% MeOH (flow of 5 ml min1). After the injection of 200 ll of sample, the collection of fractions proceeded at time intervals of 6 min, so that each run of 60 min resulted in the collection of 10 fractions. Each fraction was brought to dryness either by freeze drying or rotary evaporation, or both. Each dried residue was recovered in acetone, split in smaller and equal aliquots, and dried under nitrogen. The aliquots obtained from each fraction, resulting again in 1 l equivalent content of river pollutants, yet fractioned, were tested in vitro as the corresponding river water fractions (from 1 to 10, in the order of an increasing MeOH gradient). Following the same procedure, an analogous set of whole extract and fractions was produced starting from 10 l of unchlorinated tap water. The corresponding set of dried samples was tested in vitro as a procedural blank. A composite river sediment sample was pooled dredging at the mouth of the river Lambro before its confluence with the river Po. The thoroughly mixed sediment sample was transported to the laboratory on ice and freeze dried. Four aliquots of freeze dried sediment, each of 1.25 g, were extracted with 15 ml of MeOH in ultrasonic bath for 30 min. To remove the lipid fraction, 15 ml of hexane were added to each of them and centrifuged at 2500g for 5 min. The separated MeOH fraction was reduced to 1 ml, mixed with 9 ml of MilliQ water (adjusted to pH 3), and passed

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through Oasis HLB glass cartridges (200 mg), one for each aliquot of sediment (1.25 g). The concentrated analytes were eluted from cartridges using a mixture of 10% MeOH in tert-butyl methyl ether, pooled, and divided into two equal subsamples. According to the proceeding of the water sample, the first subsample was split into smaller equivalent aliquots which were dried under N2. Each aliquot contained the contaminants extracted from 0.5 g of the original sediment sample. It was tested in vitro as whole sediment extract. The second subsample of the sediment extract was fractioned following the same HPLC procedure described for river water. Accordingly, the aliquots finally obtained from each fraction, resulted in a contaminant content equivalent to 0.5 g of sediment, yet fractioned, and were tested in vitro as the corresponding river sediment fractions (from 1 to 10, in the order of an increasing MeOH gradient). 2.3. Assay procedure for the screening of androgenic activity The recombinant YAS was kindly provided by Prof. Sumpter, Brunel University, UK. All media used for the assay were prepared according to the original protocol for the YAS (Sohoni and Sumpter, 1998) and the original assay procedure was changed in adaptation of the improvements done for the yeast estrogen screen (De Boever et al., 2001): Addition of cycloheximide stops protein synthesis (i.e. b-galactosidase) and the combination of addition of CPRG and cycloheximide after 24 h leads to a defined incubation time of the samples with the yeast and to lower background values. Yeast (125 ll) from the yeast stock stored at 20 C were added to the growth medium and grown on an orbital shaker for about 24 h until the absorption of 1 at 620 nm was achieved. The yeast culture was diluted to an absorption of 0.1 with fresh growth medium and 150 ll were seeded into 96 well microtiter plates that were previously prepared with a dilution series of MDHT (final concentration: 7 · 108 M to 5 · 1010 M) and the samples. Ten microliters of each MDHT concentration and of each sample were transferred into microtiter plates and allowed to evaporate until dryness. The final test concentration of the water samples were 13-fold since the 1000-fold concentrated samples were previously diluted to 200-fold and finally 1:15 diluted by the yeast culture. The final test concentration of the sediment samples was 5 mg according to applied volume of the sample. The plates were sealed with wax film and vigorously shaken on a plate shaker for 2 min. After incubation for 24 h at 28 C, 50 ll of a mix of CPRG and cycloheximide were added to each well. The solution was prepared by adding 1 ml cycloheximide (20 mg ml1) and 200 ll CPRG (10 mg ml1) to 4 ml minimal medium. The plates were incubated overnight at 37 C and read at 570 nm for the colour development and at 620 nm for the turbidity with a plate reader (TECAN, SpectraFluorPlus, Crailsheim, Germany). Data were cor-

rected for turbidity as follows: corrected value = chemical absorbance at 570 nm – (chemical absorbance at 620 nm – blank absorbance at 620 nm). Untreated cells and a solvent control were included in the experimental design. At least two independent experiments were performed in triplicate for each sample. 2.4. Assay procedure for the screening of antiandrogenic activity For the screening of antiandrogenic activity a standard curve of a serial dilution of MDHT (final concentration: 7 · 108 M to 5 · 1010 M) and of FLU (final concentration: 7 · 105 M to 5 · 107 M) was transferred to microtiter plates. A parallel treatment with 6.7 · 109 M MDHT causing submaximal androgen activity was applied for the FLU standard curve and for the water and sediment samples. Again untreated cells and a solvent control were included in the experiments. At least two independent experiments were performed in triplicate for each sample. 2.5. Chemical analyses with HPLC–MS/MS and GC–MS All chemicals were purchased from Sigma Aldrich (Schnelldorf, Germany). Solvents were of HPLC grade or the best grade available and were purchased from Carlo Erba Reagenti SpA (Rodano, MI, Italy). The HPLC system consisted of two Perkin–Elmer Series 200 pumps and a Perkin–Elmer Series 200 autosampler. A LUNA C8 column 50 mm · 2 mm i.d., 5 lm particle size (Phenomenex, Torrance, CA, USA) was used for the chromatographic separation. The TIS (Turbo Ionspray) source temperature was 350 C. For the HPLC–MS/MS experiments an Applied Biosystem-SCIEX API 3000 triple quadrupole mass spectrometer was used. Mass spectrometry analyses were performed in the MRM mode (Multiple Reaction Monitoring). For the MRM experiments, the collision energy ranged from –20 to –42 eV (depending on the MRM transition) and the collision gas was nitrogen. Dried fractions and total extract were rinsed with 400 ll of MeOH. For HPLC analysis 100 ll of fractions and total extract added with the Internal standards 17b estradiold3 and Wdiethylstilbestrol-d8, methyltestosterone-d3 and C13pentachlorophenol were injected both in positive and in negative ionisation. For negative ionisation an aliquot of 20 ll of sample was mixed with 80 ll of water and 5 ng of external standard (Warfarin), 10 ll were injected in the HPLC/MRM system. For positive ionisation an aliquot of 20 ll of sample was mixed with 20 ll of acetonitrile and 60 ll of MilliQ water, adding the external standard (5 ng of Warfarin). 10 ll were injected in the HPLC/ MRM system. Different mobile phases were used depending on the ionisation and compounds analysed. For positive ionisation (compounds: ketoconazole, prochloraz, fenthion, cyproterone acetate, fenitrothion) 0.1% formic acid in MilliQ water (A) and acetonitrile (B) with a gradient from 0% to 100% B in 20 min at 200 ll min1. For

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negative ionisation (compounds: nonylphenol, BPA, tertoctylphenol, FLU) 0.05% TEA (triethylamine) in MilliQ water (A) and acetonitrile (B) with a gradient from 100% A to 100% B in 10 min at 200 ll min1. For negative ionisation (compounds: linuron, M1, iprodione) 0.05% CH3COOH in MilliQ water (A) and acetonitrile (B) with a gradient from 80% B to 20% B in 20 min at 200 ll min1. The GC–MS system was an Agilent serie 6890 gas chromatograph interfaced to an Agilent 5973 Network Mass Selective Detector HP 5971. A Zebron ZB5 30 m · 0.25 mm capillary column was used; the film thickness of the stationary phase was 0.25 lm. The injector temperature was 250 C; the column was operating with an oven temperature held at 80 C for 5 min, then programmed at 30 C min1 to 190 C and then 10 C min1 to 300 C, with a final isothermal of 300 C for 4 min. The carrier gas was helium with a constant flow of 1 ml min1. Analyses were executed in the splitless mode. The instrument was operated in the EI mode with an electron energy of 70 eV and a source temperature of 280 C. Detection was performed in SCAN and SIM mode. Dried fractions and total extract were rinsed with 400 ll of MeOH. For GC/MS analysis an aliquot of 50 ll of sample was deducted and added with the internal standard atrazin-d5. 2 ll were injected in the GC system in sim mode. Vinclozolin, vinclozolin metabolite M2, p 0 ,p 0 -DDE, dieldrin, lindane and mirex have been quantified.

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Fig. 1. Standard curves of methyldihydrotestosterone (MDHT) and of flutamide (FLU) in the yeast androgen screen (YAS). FLU data were treated in parallel with MDHT in the concentration of 6.7 · 109 M as indicated by the dashed line. The androgenic and antiandrogenic activity is shown as relative expression versus the untreated yeast cells in percent.

2.6. Statistics Data of the YAS for androgenic and antiandrogenic activity were tested for normality. Differences between the controls (solvent treatment) and the fractions of water and of sediment were tested with One-Way Analysis of Variance (ANOVA) using Dunnett’s test. Due to variance of the data in the antiandrogenic screen for the sediment samples non-parametric Dunn’s test was applied. For the calculation of the amount of androgenic and antiandrogenic activity, data were logit-log transformed and then expressed as equivalents of MDHT and FLU, respectively. 3. Results The standard curves of MDHT and of FLU in the YAS showed the general suitability of this assay to detect androgenic as well as antiandrogenic chemicals (Fig. 1). The screening for androgenic activity in the water samples from the river Lambro showed highly significant androgenic activities in fraction no. 6, 7 and 10 (p < 0.001, Fig. 2). The corresponding MDHT equivalents were 0.681 nM (207.51 ± 47.34 ng l1), 0.427 nM (130.05 ± 38.58 ng l1) and 0.499 nM (152.07 ± 23.9 ng l1), respectively. In contrast, no androgenic activity was found in the total water extract. The screenings for antiandrogenic activity in the water samples revealed antiandrogenic activities in the fraction no. 4 and 8 as well as in the total water extract

Fig. 2. Androgenic activity in the river Lambro measured with the YAS. The results for 10 water fractions (W1 to W10) and for the total water extract (Wtot.) are shown in the upper panel. In the lower panel the results for 10 sediment fractions (S1 to S10) and for the total sediment extract (Stot.) are given. The androgenic activity is expressed as relative expression versus the untreated cells (untreat.) as mean ± SD. The shown concentration of the MDHT treatment was 2 · 109 M; solvent control (EtOH) did not differ from the untreated yeast cells. Significant differences between the treatments and the control were tested using one-way analysis of variance (ANOVA, Dunnett’s test). Significant differences are indicated by asterisks (*** = p < 0.001).

(p < 0.001, Fig. 3). The corresponding FLU equivalents were 2.11 lM (583.44 ± 260.78 lg l1), 1.34 lM (369.37 ± 287.94 lg l1) and 1.59 lM (438.15 ± 188.61 lg l1), respectively. In the sediment samples from the river Lambro androgenic activity was detected in fraction no. 1, 2 and 4 (p < 0.001, Fig. 2). The corresponding MDHT

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Fig. 3. Antiandrogenic activity in the river Lambro measured with the YAS corresponding to Fig. 2. The water and sediment fractions as well as the FLU samples were treated in parallel with MDHT in the concentration of 6.7 · 109 M as indicated by the dashed line. The concentration of the MDHT treatment was 6.7 · 109 M and the concentration of FLU was 1.7 · 105 M. The arrows referred to values above saturation. Significant differences between the treatments and the MDHT treatment were tested using ANOVA, Dunnett’s test. Significant differences are indicated by asterisks (*** = p < 0.001; * = p < 0.05).

equivalents were 3.88 nM (35.4 ± 9.65 lg kg1 sediment), 2.52 nM (22.99 ± 2.12 lg kg1 sediment) and 4.38 nM (40.01 ± 14.23 lg kg1 sediment), respectively. The total sediment extract showed no androgenic activities. Antiandrogenic activity was found in the fraction no. 8 and 9 and in the total sediment extract (Fig. 3). The corresponding FLU equivalents were 5.57 lM (46.15 ± 13.84 mg kg1 sediment), 7.79 lM (64.57 ± 14.09 mg kg1 sediment) and 17.1 lM (141.73 ± 82.37 mg kg1 sediment), respectively. At the screening for antiandrogenic activities the samples were treated in parallel with MDHT at a concentration of 6.7 · 109 M. The corresponding values of water fraction no. 6, 7 and of sediment fraction no. 1, 4, 5 were above the detection limit of the photometer displaying only androgenic activities. In the figures these values are indicated by arrows. The procedural blanks of the whole extraction procedure did not display either androgenic or antiandrogenic activities. No effects of cytotoxicity were observed for any concentration of MDHT, FLU, the fractions or total extracts of water and sediment samples used in the yeast assays. The fractions and total extracts of water and sediment of the river Lambro were chemically analysed for the presence of 20 suspected antiandrogenic chemicals using HPLC– MS/MS and GC–MS analyses. Thirteen of the 20 analysed

chemicals were below the detection limit in any of the fractions or total extracts (ketoconazole, fenthion, cyproterone acetate, fenitrothion, octylphenol, FLU, pentachlorophenol, vinclozoline and the two metabolites M1 and M2, lindane, dieldrin and mirex) while seven chemicals could be detected (prochloraz, nonylphenol, BPA, tert-octylphenol, linuron, iprodione and p,p 0 -DDE). In water fraction no. 6 BPA and linuron was found with a concentration of 76.1 and 2.8 ng l1, respectively. The concentration of prochloraz was 0.4 ng l1and of iprodione 33.2 ng l1 in the water fraction no. 7 while water fraction no. 8 contained 36.5 ng l1 nonylphenol. In the total water extract nonylphenol, BPA and iprodione was detected with a concentration of 147.9, 207.1 and 91.9 ng l1, respectively. In the sediment fraction no. 8 nonylphenol was detected with a concentration of 609.5 lg kg1. In the total sediment extract nonylphenol, tert-octylphenol and the DDT metabolite p,p 0 -DDE were found with concentrations of 1428.4, 66.3 and 18.8 lg kg1, respectively. In accordance with the fractions and total extracts that showed antiandrogenic activity in the yeast assay, five of the suspected antiandrogenic chemicals were present. Nonylphenol was found in the water and sediment fraction no. 8. In the total water extract nonylphenol, BPA and iprodione were found, while in the total sediment extract nonylphenol, tert-octylphenol and p,p 0 -DDE were detected. 4. Discussion The river Lambro is a confluent to the river Po in Northern Italy. Regarded as a polluted watercourse, it is contaminated with domestic and industrial waste water as well as with agricultural run-off (Vigano et al., 1999). The load of the Lambro river water contains a complex mixture of pollutants such as pesticides (Vigano et al., 1999), trace metals (Pettine et al., 1996), organochlorines (Vigano et al., 2000) and indications for EDC since the occurrence of intersex in fish was observed (Vigano et al., 2001). The screening of water and sediment from the river Lambro for EDC revealed that the overall endocrine mode of action in the total water and sediment extract was only antiandrogenic, composed of a mixture of androgenic and antiandrogenic compounds detected by the performance of fractionation coupled YAS. The levels of androgenic activities in the river Lambro were higher than these reported from previous studies provided that equivalents of the used androgen MDHT in the present study is comparable to other androgens such as dihydrotestosterone (DHT) and R1881 used in other studies. In rivers and estuaries from the UK androgenic activities were found up to 10 ng DHT-equivalent l1, in effluents from sewage treatment plants up to 635 ng DHT-equivalent l1 and in sediments up to 15.3 lg DHT-equivalent kg1 (Thomas et al., 2002). In different surface waters of the Netherlands androgen activity up to 0.02 nmol l1 R1881 equivalents could be detected by using the AR-LUX assay but no antiandro-

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genic activity (Blankvoort et al., 2005). In the near of effluents of paper mills the degradation of phytosterols to androgens was proposed as one cause of androgenicity (Jenkins et al., 2004). Androgenic chemicals are related to masculinization (Bo¨gi et al., 2002) and depression of steroid levels and gonadal steroid biosynthesis (Sharpe et al., 2004). One wildlife study showed masculinization of mosquitofish embryos living in vicinity of a large pulp mill (Larsson and Forlin, 2002). The presence of antiandrogenic chemicals in the environment was hypothesized since the antiandrogenic potential of anthropogenic chemicals, mainly used as fungicides or herbicides, was detected. In surface water samples and effluents from industry and waste water treatment plants from the Netherlands antiandrogenic activity were detected qualitatively using monkey kidney cells (Christiaens et al., 2005). The present study reports for the first time quantified antiandrogenic activities in the environment with high levels in both water and sediment of a polluted river in Italy that ranged between 1.34 lM and 17.1 lM FLU equivalents, respectively. FLU in the lM range (400 lg l1) was shown to effectively block the trenbolone induced masculinization in fathead minnow (Ankley et al., 2004). The effects of anti-androgens have been related to reduced fertility and reproductive success of male guppies exposed to vinclozoline and p,p 0 -DDE (Baatrup and Junge, 2001; Bayley et al., 2003). The phenomenon of intersex in fish was observed downstream of the confluence of the river Lambro with significant histo-morphological alterations in about 50% of the examined gonads of barbel and was related to the river Lambro as source of EDC (Vigano et al., 2001). In laboratory studies it was shown that not only the exposure to estrogenic compounds produced feminizing effects but also the exposure to antiandrogenic compounds. In vivo exposure of Xenopus laevis tadpoles with the antiandrogen cyproterone acetate (Bo¨gi et al., 2002), vinclozoline and p,p 0 -DDE (Kloas, 2002) resulted in a significant feminization at a concentration of 108 M. In vivo exposure of adult X. laevis with Lambro river water did not show any significant effect on the mRNA expression of luteinizing hormone, follicle stimulating hormone and gonadotropin releasing hormone in the brain which would indicate disturbances of the reproductive system (Urbatzka et al., 2006). In contrast, the histo-pathological examination of the gonads of X. laevis from the same experiment exposed to Lambro river water showed a feminization effect of male gonads by the occurrence of oocytes in 20% of the males (Mandich et al., in prep.). Therefore it may be possible that the antiandrogenic charge of the water and the sediment in the river Lambro contributed to the appearance of intersex at the confluence of the river Lambro, in addition to the moderate estrogenic charge that is detailed in an accompanying paper (Van Cauwenberge et al., in prep.). The YAS as screening method based on simple eukaryotic cells clearly showed the presence of antiandrogenic compounds in the water and sediment of the river Lambro. However, it is not clear to which extent

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the results obtained with an assay based on simple eukaryotic cells can be extrapolated to complex eukaryotic organisms. Further research is needed to determine the exact load of antiandrogenic compounds in the environment and their possible health risk for aquatic living organisms. The fractions and the total extract of the water and sediment samples were examined by chemical analyses regarding the presence of chemicals with suspected antiandrogenic potency. The appearance of nonylphenol, BPA, iprodione, tert-octylphenol and p,p 0 -DDE in the fractions showing antiandrogenic activity in the YAS suggested that these compounds may have contributed to the observed antiandrogenic activities to an unknown extent. Discrepancies of mass balance between fractions and total extracts might be the consequence of the fact that fractions have passed through additional preparative steps in which there could have been some losses. In different in vitro assays these above mentioned chemicals were described to be antiandrogenic. The screening of 60 chemicals for antiandrogenic activity revealed BPA and p,p 0 -DDE as highly potent antiandrogens in a Chinese hamster ovarian cell line (Roy et al., 2004). BPA and nonylphenol displayed in vitro antiandrogenic activity by inhibiting the binding of native androgens to the androgen receptor (AR), AR nuclear localization, AR interaction with its coregulator and its subsequent transactivation (Lee et al., 2003). In contrast, in YAS nonylphenol was described as weak androgen agonist (Sohoni and Sumpter, 1998). The IC50 (inhibiting concentration 50%) value of antiandrogenic activity of tertoctylphenol and BPA was approximately 5 lM in reporter cell lines (Paris et al., 2002). Iprodione was only investigated in vivo with controversial results: in one study iprodione is a very weak antiandrogen reducing the weight of androgen dependent tissues or organs in Hershberger assay (Zhang et al., 2004) while in another study with the same assay no effect was observed (Gray et al., 1999). However, in the sediment fraction no. 9 and in the water fraction no. 4 none of the known antiandrogenic compounds were detectable but significant antiandrogenic effects were observed in YAS. These differences between the chemical analyses and the induced activities in YAS pointed to the occurrence of other yet unknown compounds that contributed clearly to the observed antiandrogenic activity in the water and sediment of the river Lambro. Some of the suspected antiandrogenic compounds that were also present in fractions and extracts of Lambro samples (BPA, nonylphenol, p,p 0 -DDE) have been previously tested for their (anti)androgenic activity in YAS (Sohoni and Sumpter, 1998). Antiandrogenic response was observed in YAS in the 4–6 lM range for BPA and p, p 0 -DDE while androgenic response was in the lM range for nonylphenol and in the 10 lM range for p,p 0 -DDE. Neither BPA, nonylphenol nor p,p 0 -DDE were present in the lM range in the fractions or extracts of the river Lambro but in the nM range or lower as revealed by chemical analysis. This supports the hypothesis that mainly still unknown compounds produced the antiandrogenic responses in samples from the river Lambro.

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In conclusion, in vitro cell based assays in combination with a fractionation procedure and chemical analyses are very useful to investigate environmental samples containing mixtures of known and unknown chemical compounds with different modes of actions. The screening of water and sediment samples of the river Lambro revealed androgenic and antiandrogenic activities in the fractions but solely antiandrogenic activity in the total extracts. The combination of the YAS with the chemical analyses showed that mainly unknown compounds but may be in part nonylphenol, BPA, iprodione, tert-octylphenol and p,p 0 -DDE in an unknown extent are responsible for the observed overall antiandrogenic activity. This is the first study quantifying antiandrogenic activities in environmental samples in combination with chemical analysis to detect the origin of antiandrogenic activities. The presence of antiandrogens in the river Lambro provides evidence that the screening of EDC in the environment that have the potential to interfere with reproduction of aquatic organisms should be extended to all modes of action including androgenic and antiandrogenic ones and that besides some moderate estrogenic activities an important source of EDC at least in the river Lambro possesses antiandrogenic activity. Acknowledgement This research was funded by the European Union within the EU-project EASYRING, contract No. QLK4-CT2002-02286. References Ankley, G.T., Defoe, D.L., Kahl, M.D., Jensen, K.M., Makynen, E.A., Miracle, A., Hartig, P., Gray, L.E., Cardon, M., Wilson, V., 2004. Evaluation of the model anti-androgen flutamide for assessing the mechanistic basis of responses to an androgen in the fathead minnow (Pimephales promelas). Environ. Sci. Technol. 38, 6322–6327. Baatrup, E., Junge, M., 2001. Antiandrogenic pesticides disrupt sexual characteristics in the adult male guppy Poecilia reticulata. Environ. Health Persp. 109, 1063–1070. Bayley, M., Larsen, P.F., Baekgaard, H., Baatrup, E., 2003. The effects of vinclozolin, an anti-androgenic fungicide, on male guppy secondary sex characters and reproductive success. Biol. Reprod. 69, 1951–1956. Blankvoort, B.M.G., Rodenburg, R.J.T., Murk, A.J., Koeman, J.H., Schilt, R., Aarts, J.M.M.J., 2005. Androgenic activity in surface water samples detected using the AR-LUX assay: indications for mixture effects. Environ. Toxicol. Phar. 19, 263–272. Bo¨gi, C., Levy, G., Lutz, I., Kloas, W., 2002. Functional genomics and sexual differentiation in amphibians. Comp. Biochem. Phys. B 133, 559–570. Cargouet, M., Perdiz, D., Mouatassim-Souali, A., Tamisier-Karolak, S., Levi, Y., 2004. Assessment of river contamination by estrogenic compounds in Paris area (France). Sci. Total Environ. 324, 55–66. Christiaens, V., Berckmans, P., Haelens, A., Witters, H., Claessens, F., 2005. Comparison of different androgen bioassays in the screening for environmental (anti)androgenic activity. Environ. Toxicol. Chem. 24, 2646–2656. De Boever, P., Demare, W., Vanderperren, E., Cooreman, K., Bossier, P., Verstraete, W., 2001. Optimization of a yeast estrogen screen and its applicability to study the release of estrogenic isoflavones from a soygerm powder. Environ. Health Persp. 109, 691–697.

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